The effects of CO2 and external buffering on ammonia excretion and Rhesus glycoprotein mRNA expression in rainbow trout

Rhesus (Rh) proteins were recently characterized as ammonia gas (NH3) channels. Studies indicate, however, that Rh proteins also facilitate CO2 transport in a green alga and in human erythrocytes. Previously, we reported that Rh mRNA expression in various rainbow trout tissues responded to high environmental ammonia. To determine whether or not Rh proteins may also be involved in CO2 transport in rainbow trout, we examined the effects of a 12 h exposure to external hypercapnia (1% CO2 in air) on Rh mRNA expression in the gill, skin and erythrocytes. External hypercapnic conditions lowered the water pH and facilitated ammonia excretion; therefore, we also studied the effects of hypercapnia and normocapnia in the presence of 10 mmol l(-1) Hepes-buffered water. Hepes treatment prevented water acidification, but resulted in elevated plasma ammonia levels and reduced ammonia excretion rates. Hypercapnia exposure without buffering did not elicit changes in Rh mRNA expression in the gill or skin. However, Rhcg2 mRNA expression was downregulated in the gills and upregulated in the skin of both normocapnia- and hypercapnia-exposed fish in Hepes-buffered water. mRNA expression of a newly cloned Rhbg2 cDNA was downregulated in the skin of fish exposed to buffered water, and Rhag mRNA expression in erythrocytes was decreased with exposure to normocapnia in buffered water but not with hypercapnia exposure in either buffered or unbuffered water. With the aid of Hepes buffering, we were able to observe the effects of both CO2 and ammonia on Rh mRNA expression. Overall, we conclude that high CO2 did not directly elicit changes in Rh mRNA transcription levels in the gill and skin, and that the changes observed probably reflect responses to high plasma ammonia, mirroring those in trout exposed to high environmental ammonia. Therefore a dual function for gill and skin Rh proteins in CO2 and ammonia transport is not evident from these results. Rhag expression, however, responded differentially to high CO2 and high ammonia, suggesting a possible dual role in the erythrocytes.